Prion diseases, which are also called transmissible spongiform encephalopathies (TSEs), include a group of fatal infectious neurodegenerative diseases that include Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler Scheinker syndrome (GSS), fatal familial insomnia (FFI) and sporadic fatal insomnia (sFI) in humans, and scrapie, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in animals. These diseases are characterized by brain vacuolation, astrogliosis, neuronal apoptosis, and the accumulation of misfolded prion protein (PrP-res, also known as PrPSc and PrPCJD) in the central nervous system. TSEs have incubation periods of months to years, but after the appearance of clinical signs they are rapidly progressive, untreatable, and invariably fatal. Attempts at TSE risk reduction have led to profound changes in the production and trade of agricultural goods, medicines, cosmetics, and biotechnology products.
The hallmark event of prion disease is the formation of an abnormally folded protein called PrPSc (or PrP-res), which is a post-translationally modified version of a normal protein, termed PrPC (also known as PrP-sen). A prion detection method termed protein misfolding cyclic amplification (PMCA) is based on the ability of prions to replicate in vitro in cell lysates containing PrPC (see, for instance, WO0204954). However, the limitations of PMCA include the time required to achieve optimal sensitivity (˜3 weeks) and the requirement for brain-derived PrP-sen as the amplification substrate.
Castilla et al., Methods in Enzymology 412:3-21 (2006) has stated that it has not been possible to use PMCA with highly purified prion proteins such as PrPC. Although the reason for this limitation was unknown, it was believed that factors in brain homogenates were needed to catalyze prion propagation. Recombinant PrP-sen expressed from E. coli also lacks glycosylation and the glycophosphatidylinositol (GPI) anchor, which was additionally believed to contribute to the difficulty of using rPrP-sen in amplification reactions. Such rPrP-sen has been converted to protease-resistant forms with very limited yields when mixed with PrPSc in the past.
Another problem with PMCA is that the formation of PrPSc reaches a plateau as the number of amplification cycles increases. Castrillon et al. (US Patent Publication No. 2006/0263767) attempted to overcome this problem by serial amplification of prion protein by removing a portion of the reaction mix and incubating it with additional non-pathogenic protein. Although serial amplification PMCA (saPMCA) increases prion amplification and enhances the sensitivity of the assay, the necessity of performing multiple rounds of serial amplification has decreased the overall practicality of the process.
Supattapone and Deleault (PCT Publication No. WO 2007/082173) also note that efficiency of amplification may require a cellular factor other than PrP-sen. They disclose in vitro amplification of immunoaffinity or exchange chromatography purified PrP-sen in the presence of RNA, synthetic polyanions and partially purified substrates to increase the sensitivity of diagnostic methods for detecting PrP-res.
However, there continues to be a need for a more rapid method for the detection of PrP-res that is sensitive enough to detect low level prion contamination. The widespread public health concern about TSE diseases could be allayed by the development of such a test.